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Cytogenetics

Monitor & Document Cell Growth

Once cultures are established, the laboratory enters the surveillance phase. The objective is to maintain cells in the exponential growth phase, protect them from biological invaders, and accurately identify the optimal moment for harvest. Continuous monitoring allows for the early detection of failures, distinguishing between salvageable technical errors and irreversible specimen limitations

Detect, Identify, & Control Contamination

Contamination is the greatest threat to culture viability. Early detection prevents the spread of pathogens to other patient samples

  • Detection (Visual Cues)
    • Turbidity: Cloudiness indicates bacterial overgrowth
    • pH Shift: Rapid Yellowing of media (acidic) signals bacterial metabolism; Purple (alkaline) may indicate fungal or yeast activity
    • Microscopy: “Shimmering” dots (bacteria) or branching filaments (fungi) visible between cells
  • Identification
    • Bacteria: Fast-growing (24hr), typically skin flora (Staph/Strep) from technique breach
    • Fungi: Slow-growing “cotton balls,” often from the environment/incubator humidity pans
    • Mycoplasma: Invisible to the naked eye/light microscope; requires PCR testing. Causes chromosomal damage without killing the culture
  • Control
    • Isolation: Remove suspicious vessels immediately. Do not open fungal cultures in the lab
    • Remediation: “Wash and Rescue” with heavy antibiotics (Gentamicin/Nystatin) can sometimes save critical specimens (marrow/amnio), but discarded is the norm for replaceable samples (blood)

Culture Maintenance

Cells require regular intervention to replenish nutrients and dilute toxic metabolites

  • Feeding
    • Suspension: Often unnecessary for short-term (72hr) cultures
    • Monolayer: Media is changed every 2–3 days. Partial changes (50%) retain beneficial paracrine growth factors
  • Environment: Daily checks of Temperature (\(37.0^\circ\text{C}\)), CO2 (5%), and Humidity (\(>90\%\)) are mandatory. Equipment failure (e.g., dry water pans) leads to hypertonic shock and cell death

Evaluate/Subculture Monolayer Cells

Anchorage-dependent cells (Amnio/Tissue) must be managed based on density to prevent contact inhibition

  • Evaluation: Using an inverted microscope to assess Confluence (coverage %) and Morphology (health)
    • Log Phase (50–80%): Optimal for harvest. Dividing cells are round/refractile
    • Confluent (100%): Contact Inhibition: stops division. Yields zero metaphases
  • Subculturing (Passaging)
    • Trypsinization: Enzymatic detachment of cells when they approach confluence (but aren’t ready for harvest). Cells are split 1:2 or 1:3 into new flasks
    • Protocol: Rinse with saline (remove serum inhibitor) \(\rightarrow\) Trypsin digest \(\rightarrow\) Neutralize with Serum \(\rightarrow\) Reseed
    • Amnio Rescue: “Cleaning up” bloody samples by changing media at 24hrs to remove RBCs while retaining adherent amniocytes

Assess Cultures for Harvest

The decision to add Colcemid is the “Point of No Return.”

  • Suspension (Blood): Time-dependent. Harvested at 72 hours (peak PHA stimulation) regardless of visual density
  • Monolayer (Amnio/Tissue): Density-dependent. Harvest targeted at 60–80% confluence with visible mitotic figures (round cells). Overgrown cultures must be split, not harvested
  • In Situ Amnio: Requires 15+ independent colonies. Dishes are harvested sequentially (not all at once) to provide backup

Investigate & Document Culture Failures

Every failure is a sentinel event requiring classification

  • Biological Failure (Specimen-Related)
    • Low viability (transport delay, heat exposure), low cellularity (dry tap marrow), or necrotic tissue. Unpreventable by the lab. Reported as “Unsuccessful Study” with a description of the specimen condition
  • Technical Failure (Lab-Related)
    • Incubator malfunction, contaminated media lots, or processing errors (forgetting PHA). Investigated via QA audit (e.g., checking if all cultures from “Lot X” failed)
  • Documentation: Failures are logged in the QA database for “Root Cause Analysis.” If a sample fails, the physician is notified immediately to allow for recollection